Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Fluorescent dyes are used to impart both visible color and fluorescence to other materials. Particularly useful are fluorescent reagents that exhibit selectivity in their labeling reactions, permitting the detection and/or identification of particular substances, or identification of characteristics of a sample.
A variety of detectable hydrazine, hydroxylamine and amine derivatives have been described that share the utility of labeling aldehyde and ketone functional groups. Among the most widely used of such reagents are dansyl hydrazine, fluorescein thiosemicarbazide, various biotin hydrazides, biotin hydroxylamine (ARP), and various aromatic amines (2-aminopyridine, 8-aminonaphthalene-1,3,6-disulfonic acid, 1-aminopyrene-3,6,8-trisulfonic acid, 2-aminoacridone) (as described by Haugland et al. MOLECULAR PROBES, INC. HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 7TH EDITION, on CD-ROM, Chapter 3, and references cited therein, which are incorporated by reference).
Most existing methods of labeling carbohydrates that utilize hydrazine, hydroxylamine and amine derivatization reagents have focused on labeling aldehydes present in, or introduced into, carbohydrates, particularly the so-called “reducing sugars”. The adduct formed with the reducing sugar can be further stabilized by treatment with borohydride or a cyanoborohydride. The derivatization reaction typically proceeds or is followed by a separation technique such as chromatography, electrophoresis, precipitation, affinity isolation or other means before direct or indirect detection of the labeled product.
In addition, some simple and complex sugars have been derivatized by coupling to a carboxylic acid, as in glucuronides. Typically, the amine-, hydrazine- or hydroxylamine-substituted label functions as a nucleophile, and the carboxylic acid must first be activated by forming an ester or an anhydride, using an activating agent such as a carbodiimide (for example ethyldimethylaminopropyl carbodiimide, or EDAC). Capillary electrophoresis has been particularly effective for analysis of adducts or detectable derivatives with carbohydrates, including complex carbohydrates and carbohydrates obtained by hydrolysis of glycoproteins, gangliosides and other sugar-containing biomoleules. The detectable adducts thus formed in each case with the sugar or polysaccharide can themselves be utilized as tracers, for receptor binding, as enzyme substrates and for multiple other applications.
The derivatization reagents of this invention show fluorescence properties superior to those of any similar reagents that have been described for detection of aldehyde- or ketone-containing molecules in gels, and permit bright fluorescent labeling of carbohydrates, glycoproteins, glycogen, lipopolysaccharides, and other aldehyde, ketone, carboxylic acid, or sulfonic acid containing substances. Unlike biotin hydrazides, biotin hydroxylamines and digoxigenin hydrazides, all of which require blotting onto a membrane and the use of a secondary detection reagent, the reagents of the invention permit rapid detection of oxidized glycoproteins within gels.
The preferred reagents of the invention are well excited by ultraviolet excitation sources commonly used in epi-illuminators and transilluminators. The dyes possess unusually high Stokes shifts of greater than about 150 nm, effectively reducing background due to autofluorescence and scattering of the exciting light. In addition, the staining procedure of this invention is rapid and mild, exhibits substantially greater sensitivity than previously utilized labeling reagents, and employs a simple staining procedure.
A family of fluorogenic substrates that yield highly fluorescent products and that are related to the reagents of the invention has been previously described (U.S. Pat. No. 5,316,906 to Haugland et al. (1994); U.S. Pat. No. 5,443,986 to Haugland et al. (1995); both incorporated by reference). However, these known fluorogenic substrates are nonfluorescent until enzyme action, are not substituted by a reactive functional group to label a desired target substance, and their fluorescent products are designed to be insoluble under physiological conditions. In contrast, in preferred embodiments of the invention, the non-fluorescent reagents only become fluorescent when bound to the biomolecules with aldehyde or ketone moieties, e.g. in a gel or on a membrane.